Method of identification and cloning differentially expressed messenger RNAs

ABSTRACT

A method of identification of differentially expressed messenger RNA (mRNA) which consists of synthesizing from a set of sequences of mRNA sets of fragments of complementary DNA (cDNA), which are separated with the aid of gel electrophoresis and the pictures of separation of the cDNA from different types of cells are compared and fragments with differential signal intensity are identified. For formation of the set of fragments the cDNA is cleaved with the aid of restriction nucleases. A method of cloning of differentially expressed mRNAs consists of synthesizing from sets of sequences of mRNAs from different types of cells sets of fragments of complementary DNA (cDNA) which are separated with the aid of gel electrophoresis, the pictures of the separation of the cDNA from different types of cells are compared, fragments of cDNA with different signal intensities are separated from the gel, amplified with the aid of a polymerase chain reaction and cloned to a plasmid or phage vector. For the formation of the set of fragments one carries out cleavage of the cDNA with the aid of restriction endonucleases and uses only those fragments of cDNA that correspond to the 3′ or 5′ end regions of the mRNAs.

ART

[0001] This invention relates to the field of molecular biology and canbe used in medicine and molecular biology for analysis of geneexpression and diagnosis and identification of mechanisms of pathologyat the genetic level.

PRIOR ART

[0002] Differential expression of genes, which is realized via thesynthesis of various sets of messenger RNAs (mRNAs), is the basis of thevariety of phenotypes and functions of the cells of living organisms. Inthis connection one can understand the importance of methods that mightallow identification and investigation of differentially expressedmRNAs, i.e., those RNAs, whose concentration in the cellular mRNA pooldiffers in two or more types of cells or changes in dependence on thefunctional state of the cells.

[0003] A number of methods have been described for the detection andcloning of differentially expressed mRNAs. In most cases they come downto the use of the method of hybridization of nucleic acids. In thedifferential screening method (see literature [1]) by means of transferto nitrocellulose or nylon filters replicas of libraries ofcomplementary DNA (cDNA) from the population of cells A sown in dishesare hybridized alternately with labeled preparations of cDNA from thepopulation of cells A, B, C and so forth. Since the hybridization signalobtained from individual clones theoretically should be proportional tothe representation of the corresponding cloned sequence in the cDNApreparation, one can, from the level of the signal obtained fromdifferent preparations, identify clones corresponding to genes that areexpressed differentially in the populations of cells A, B and so forth.An important shortcoming of this approach is its low sensitivity, sinceonly highly represented sequences (as a rule, those comprising at least0.1% mRNA) can be detected. For less common sequences the hybridizationsignal does not exceed the background. In addition, in this approach onecan examine only a small number (from several thousands to several tensof thousands) of clones. Another shortcoming is that information aboutthe individuality of the clone (the description of its location in thedish or replica) is lost at the end of work with these replicas andcannot be used in subsequent experiments. Moreover, cDNA libraries fromdifferent types of cells are not comparable in that identical sequencesoccupy different places on filters in different libraries. Thus,conversion from one type of cells to other types is essentiallyimpossible. Recently attempts have been made to create ordered cDNAlibraries containing individual clones distributed in the cells of96-well planchets. However, this does not eliminate the principalshortcoming of low sensitivity. In addition, sets of clones obtainedfrom one type of cell are still not comparable to sets of clones fromanother type of cell.

[0004] The method of subtracted hybridization is used in order toovercome the low sensitivity of the differential screening method [1];in this method a cDNA preparation from cells A is hybridized with a cDNAor mRNA preparation from cells B, after which the resulting hybrids areremoved by one or another method. The resulting preparation is rich insequences that are specific for preparation A. This “subtracted”preparation can be used both for the creation of libraries and fordifferential screening, since it makes it possible both to increase theprobability of finding differentially expressed sequences and toincrease the sensitivity of detection of poorly represented sequences.However, the use of this procedure makes the approach lengthy andlaborious and, in addition, the gain in sensitivity is stillinsufficient for detection of rare sequences and is accompanied by theloss of the possibility of making a direct comparison of the level ofgene expression in different cells.

[0005] Recently a modification of the differential screening method wasproposed, in which instead of cloned sequences large orderly arrangedsets of oligonucleotides are used for immobilization on a solidsubstrate, for example, all possible tetradecanenucleotides containingthe common sequence AATAAA, which is encountered in most mRNAs [2]. Thismethod theoretically provides an exhaustive search of a much larger setof mRNA sequences than in standard differential screening. Theshortcoming of this approach, like other differential screening methods,is that under conditions of hybridization with an excessively complexmixture of cDNA molecules, the frequencies of representation of whichmay vary by factors of hundreds and thousands, the probability of theappearance of false signals owing to cross hybridization becomessignificant, which makes reliable detection and quantitative analysis ofrare mRNA sequences problematical. An additional problem is the need forsynthesis and immobilization on a small area of from several dozens tohundreds of thousands of oligonucleotides, which requires a largeinitial investment. In addition, the approach in and of itself does notprovide the possibility of cloning any long segments of differentiallyexpressed mRNAs, which requires a separate operation.

[0006] Closest to the present invention is a method which is called “PCRmated with reverse transcription, with arbitrary primers” (arbitraryprimed RT-PCR). In this method cDNA synthesized by mRNA is used tocreate a set of cDNA fragments of discrete length with the aid of apolymerase chain reaction (PCR) using primers of a random sequence undernonrigorous conditions which contribute to priming and amplification notof one, as is usual in standard PCR, but rather of a whole group of cDNAsequences. The set of discrete cDNA fragments is separated byelectrophoresis in acrylamide gel and compared with a set of fragmentsobtained under identical conditions from a different type of cell. Byusing many arbitrary primers, one can compare one or several discretecDNA fragments to most mRNA species. Two principal modifications of thisapproach have been proposed. One of them uses one or two “arbitrary”primers [3,4]. The second approach uses one arbitrary and one oligo (dT)primer that contains two additional bases at the 3′ end [5,6,7]. In thiscase the amplified segments are adjacent to the 3′ and (poly(A) tail) ofthe mRNA. An important shortcoming of this approach is the fact that nomore than a few dozen sequences are amplified in a single reaction.Thus, for an exhaustive search of 10-15 thousand RNA sequences expressedin individual types of cells or tissues, as a minimum several hundred(probably several thousand) reactions must be carried out. In addition,the sampling of sequences amplified in one reaction is random and,therefore, in the exhaustive search of a significant portion ofsequences of cellular mRNA there will be a very significant accumulationof excess information. Thus, after a survey of 50% of all sequences, ineach subsequent reaction only {fraction (1/2)} of the amplifiedsequences will be now. At a depth of review of 90% the fraction of nowsequences will fall to {fraction (1/10)}. Probably the most significantshortcoming of the approach is its poor reproducibility, which isconnected with the fact that under weakly selective priming conditionssmall variations in the starting conditions or the quality of the RNApreparations, primers and other parameters will cause significantquantitative changes in the spectrum of amplified cDNA fragments. Thus,if the optimum annealing temperature is 42° C. a shift of the annealingtemperature up or down by 2° resulted in the appearance of a substantialbackground (i.e., additional bands) or to disappearance of some bands[7]. Second, according to the data of the authors of [7] when parallelexperiments were carried out, about 95% of bands proved to bereproducible [6]. Thus, the level of nonreproducibility (5%) iscomparable to or even exceeds the level of differences between mRNApopulations. According to our data, when this approach is used, thenumber of reproducible differential bands is usually smaller than thenumber of nonreproducible bands. It is probable that the sensitivity ofthe approach to the quality of preparations of RNA, primers, annealingtime and other parameters may substantially prevent a comparison ofresults with known data.

[0007] Essence of Invention

[0008] The basis of the invention was the task of supporting thepossibility of a direct qualitative and quantitative comparison of thespectra of mRNAs synthesized in different types of cells of the sameorganism, the possibility of detection of differentially expressed RNAs,including those with a low level of representation, the possibility ofcorrelating these RNAs with known ones, and also the possibility ofcloning fragments of such RNAs. The problem is resolved by the fact thatthe set of messenger RNAs expressed in a cell by means of synthesis ofcomplementary DNA (cDNA) with subsequent fragmentation of the cDNA byfrequently cleaving restriction endonucleases (having four- orfive-letter recognition sites) and use of the cDNA fragmentscorresponding to the 3′ or 5′ ends of the mRNA, is represented in theform of a set of fragments of cDNA of discrete length, no more than onefragment for each species of mRNA, and at least one of the ends of thefragments carries the marker or group necessary for detection. Thegenerated fragments of cDNA originate from the 3′ end (adjacent to thepoly(A) tail) or 5′ end regions of the mRNAs and support representation,depending on the specific embodiment of the invention, of from 90% toessentially 100% of mRNA sequences. For an increase of the sensitivityof the approach and for unambiguous identification of individualfragments the set of cDNA fragments may be divided into severalnonintersecting subsets of fragments. The sets or subsets are divided byone- or two-dimensional gel electrophoresis and detection of the markerand comparison of the separation pictures is carried out. In this casethe intensity of the signal from each fragment of cDNA varies for mRNApreparations from different cells in proportion to the representation ofthe sequence corresponding to it in the mRNA pool. If necessary in thedetection stage, by means of transfer of the separated fragments to amembrane and sequential hybridization with a set of oligonucleotides,which partially overlap the common end sequence of the fragments,additional analysis of the separation picture is possible. The separatedcDNA fragments, after additional operations can be amplified andinvestigated by means of restriction and hybridization analysis and alsosequencing, and can be cloned to a plasmid or phage vector.

[0009] Thus, in the proposed invention, in contrast to the approachusing arbitrary primers, at least one of the boundaries of the fragmentsis specified by the position of the restriction site*; in addition, inthe proposed approach a set of cDNA fragments that is as representativeas possible is created at first and then divided into nonintersectingsubsets and than separated with the aid of gel electrophoresis. The useof the reaction of cleavage of DNA by restriction nucleases, which ischaracterized by high specificity, completeness of cleavage and lowsensitivity to variations of temperature conditions, makes it possibleto make the approach much more reproducible and, therefore, will makepossible a reliable comparison with independently conducted experiments.These characteristics, together with the high resolution of the method,will make it possible to begin the creation of databases of gelcoordinates of expressed sequences. The proposed method makes itpossible to eliminate the excess of information that is characteristicfor the method with arbitrary primers. In addition the presence of onecommon population of cDNA fragments in the initial stages makes itpossible to conduct such operations as frequency equalizations by meansof self-hybridization of the population of cDNA fragments [8], whichwill make it possible to reduce substantially the level ofrepresentation of the most frequently encountered mRNA species and willhelp the isolation of very rare mRNA sequences owing to a reduction ofinterference from frequently encountered sequences.

LIST OF FIGURES

[0010]FIG. 1 shows one version of implementing the invention by means ofthe formation of a set of 3′ and labeled fragments of cDNA, dividing itinto subsets of fragments with the aid of immobilization on a solidsupport and sequential treatment with a series of restriction nucleases,and separation of the resulting subsets by electrophoresis.

[0011]FIG. 2 shown a scheme of amplification and cloning of cDNAfragments obtained and separated in gel with the aid of the method shownin FIG. 1.

[0012]FIG. 3 shows a picture of separation in denaturing polyacrylamidegel electrophoresis of ³²P-labeled fragments of cDNA from preparationsof RNA of mouse thymus (a) and mouse spleen (b) obtained with the aid ofthe method presented in FIG. 1. The first cleavage was done with theenzymes Sau3A (2-5) and BamHI (6), the second with the enzymes EcoRV(2), PstI (3, 6), mspI (4) and Hin PI (5). Microfragments of DNA areplotted on the left (1) and their lengths in nucleotides are indicated.

[0013]FIG. 4 represents hybridization of cloned fragment 1 (a) andfragment 2 (b) with preparations of poly(A)⁺ RNA from mouse spleen (1)and thymus (2) separated by electrophoresis and transferred to amembrane. The picture of hybridization confirms the specificity ofexpression that follows from FIG. 3. On the right are plotted markersingle-chain DNA fragments and their lengths and nucleotides areindicated.

[0014]FIG. 5 shows an alternative method of dividing the set of 3′ andfragments of cDNA obtained by the method shown in FIG. 1 into foursubsets by means of amplification of the set of fragments by apolymerase chain reaction with adaptor primers that contain asupplemental base at the 3′ end beyond the limits of the part that iscommon for all fragments.

[0015]FIG. 6 shows a method of creating a set of 5′ end fragments ofcDNA.

[0016] One variation of the announced method is presented in more detailbelow.

[0017] Synthesis of the first chain of cDNA is accomplished by theenzymatic route with the aid of reverse transcriptase and oligo(dT)primer immobilized on microgranular solid support or ofoligo(dT)-containing primer carrying a biotin group at the 5′ end. Forsynthesis of the second chain of cDNA, preliminary suspension of theoligo(dG) tail at the 3′ end of the first chain of cDNA with the aid ofterminal transferase is carried out. Synthesis of the second chain iscarried out with modified DNA polymerase of phage T7 sequenase (UnitedStates Biochemicals, USA) using an oligo(dC)-containing primer asprimer. Complete hydrolysis of the cDNA with restriction endonucloasehaving a four-letter recognition site (for example, Sau3A), rinsing ofthe released cleavage products and ligation of the adaptor complementaryto the cleavage site are carried out. If a biotinylized primer is usedfor synthesis of the first chain, the cDNA before or after cleavage withrestrictase is bonded to a streptavidin-containing solid support(Streptavidin MagneSphere, Promega, USA). The ability of streptavidin tobind the biotin group rapidly and stably is used in this operation. Toincrease the above material, amplification of the fragments of cDNA iscarried out by means of PCR using the primer that is in the adaptor andmodified oligo(dT) primer containing the biotin group at the 5′ end.After immobilization of the fragments in the streptavidin-containingsolid support the free ends are removed by treating with 100 mM NaOH,and then the primer is annealed in the immobilized chains, the label isadded to the 3′ end of the primer by the chain lengthening reaction withDNA polymerase using radioactive α-³²P or α-³³P DATP, after whichunlabeled deoxynucleotide triphosphates are added and complete chains ofcDNA are completed. The sequence of primer and segment cDNA annealedwith it and also the conditions of the labeling reaction are such thatthe region labeled is limited to the adaptor sequence. After adding themarker, sequential exhaustive cleavage with 8-10 restrictases is carriedout, with restrictases having 6-letter recognition sites being usedfirst, followed by 5-letter and finally 4-letter recognition sites. Thereleased labeled fragments of cDNA after each reaction are collectedseparately. Separation of the fragments is done with one of the systemsfor electrophoresis in polyacrylamide or similar gal (Hydrolink, ATBiochem, USA). The system of two-dimensional electrophoresis of DNA, inwhich separation of the double-chain DNA along the length is carried outin the first reaction, and separation with regard to composition is donein the second direction (more precisely, over the melting profile) witha denaturing gradient [9]. According to literature data, even aftertransfer to a membrane, which significantly degrades resolution, thiskind of system makes it possible to separate at least 625 fragments ofDNA on one gel [10]. To all appearances the upper limit of resolution is1 to 1.5 thousand DNA fragments. If the population of cDNA fragments isdivided into 8-12 nonintersecting sets, one can separate up to 10-15thousand fragments, i.e., essentially all mRNAs expressed in cells of asingle type. The signal from the separated fragments is detected bymeans of autoradiography on x-ray film. The pictures obtained frompreparations of RNA from different types of cells are compared with eachother, the correspondence between individual fragments of cDNA fromdifferent cells is established from the position in one- ortwo-dimensional electrophoresis and fragments giving a differentialsignal are identified.

[0018] In order to ascertain the sequence of known sequences to whichone or another spot on the two-dimensional electrophoreogram maycorrespond, one first, based on cloned sequences of mRNAs in the databank, determines which sequences may give fragments of the correspondinglink and then, by using the algorithm for prediction of mobilities offragments of DNA in a denaturing gradient [11], one can chose from thosesequences the one whose melting profile most accurately corresponds tothe position occupied by the fragment in the second direction.

[0019] In order to obtain the needed fragment in a quantity sufficientfor analysis and cloning, the fragment is eluted from the gel, theoligo(dG) tail is suspended with the aid of terminal transferase, andthen amplified by means of PCR using adaptor primer and oligo(dC) (FIG.2). The identical nature of the fragment of the known sequence can beverified by means of restriction analysis or by determining thenucleotide sequence directly in the amplified fragment. In addition, theamplified fragment may be easily cloned to the plasmid or phage vector.

[0020] Our computer analysis of 659 cloned sequences of mouse mRNAhaving a sequestered region belonging to the poly(A) tail showed thatwhen the restriction endonuclease Sau3A (recognition site: GATC) wasused for cleavage of the cDNA 93% of all mRNAs will be cleaved by theenzyme and, therefore, will participate in the analysis. The fraction ofsequences of mRNAs participating in the analysis can be increased to99%-99.5%, if cleavage is done with a second restrictase having adifferent recognition site of those sequences of cDNA that were notcleaved by the first restrictase. The computer analysis also shows thatsecond cleavage of immobilized fragments of cDNA with a set ofrestriction endonucleases can reach the overwhelming majority offragments (96% if tan four-letter restrictases are used). Thus, from 90to 96% of all mRNA sequences of the cell will be represented in the formof discrete fragments of cDNAs and will participate in the analysisperiod.

[0021] The proposed method has high sensitivity. Using only 1 μg of cDNAfragments for labeling it is easy to obtain inclusion to 10⁸ count/min.This level of labeling makes it possible to detect mRNA sequences thatmake up from 0.001% to 0.0001% of the mRNAs of the cell.

[0022] Besides the variation described above, there are also othervariations of the invention. Synthesis of the second chain of cDNA canbe accomplished by other known methods by using as primers for synthesisthe chain or RNA in hybrid cDNA-RNA that has been cleaved with the aidof RNAse H or by using self-priming of the first cDNA chain afterhydrolysis of the RNA chain [12]. The most important modification is theuse of the alternative method of dividing the set of 3′ end fragments ofcDNA into nonintersecting subsets of fragments by means of amplificationwith the aid of 12 different pairs of primers, which include a) fourversions of a modified adaptor primer, which includes an adaptor endsequence that is common for all fragments of cDNA and that each containsone additional base at the 3′ end; b) three versions of oligo(dT)containing primer that contains one additional bass at the 3′ end. Inthis case one employs the property of Taq DNA polymerase to extend onlythose primers that contain a completely paired 3′ and base [13]. In thisversion it is not necessary to carry out second cleavage with the set ofrestrictases and, therefore, the share of mRNA sequences thatparticipate in the analysis can essentially reach 100%.

[0023] One further important version of the approach is creation of aset of fragments of cDNAs corresponding to the 5′ end of the mRNAs (FIG.6). For this one uses specific labeling of the 5′ end of the mRNAs withan oligonucleotide primer (primer 1) in accordance with the procedure of[14]. After this, one synthesizes the first chain with the aid of a setof random haxamer primers, synthesizes the second chain with the aid ofprimer 1 containing a biotin group at the 5′ end, immobilizes the cDNAsin streptavidin microgranules, followed by hydrolysis of the cDNAs withrestrictase, ligation of the adaptor and other procedures, as indicatedabove. Dividing the set of 5′ end fragments of cDNAs into subsets isaccomplished either by immobilization of the labeled fragments andsequences by treatment with restrictases or with the aid of separatereactions of amplifications with and primers that contain an additionalbase at the 3′ end, as indicated above.

[0024] Besides two-dimensional electrophoresis using a denaturinggradient in the second direction, it is also possible to use systems oftwo-dimensional electrophoresis that use in the second directionseparation of single-chain DNA under nondenaturing conditions owing itsconformational polymorphism [15] or cleavage of DNA after the firstdirection by frequently cleaving restrictases [16] and separation bylength in the second direction.

[0025] Besides the use of radioactive ³²P or ³³P markers with subsequentautoradiography for detection, a nonradioactive variation of detectionmay also be accomplished; in this version labeling of the fragments ofcDNA is done with biotin groups or other chemical groups that aftertransfer of the fragments to the membrane and immobilization aredetected with one of the commercial systems of nonradioactive detectionusing chemiluminescence.

[0026] Detection of the separated unlabeled elements of cDNA can be doneby transfer of fragments to a membrane and hybridization with a labeledadaptor primer. In this case, after hybridization with the adaptorprimers that contain additional basis at the 3′ and, selectedvisualization of the subsets of fragments separated in the given gel ispossible.

[0027] Information Confirming the Possibility of Implementation of theInvention

[0028] To support the possibility of identification and cloning ofdifferentially expressed mRNAs, the experiments presented in thefollowing example were carried out.

EXAMPLE

[0029] Identification and cloning of mRNAs differentially expressed inmouse thymus and spleen.

[0030] Preparation of total RNA are extracted from mouse thymus andspleen in parallel using extraction with acid phenol [17]. Synthesis ofthe first cDNA chain was accomplished under the following conditions:37° C., 60 min, reaction volume 20 μL, 5 μg total RNA, 200 U reversetranscriptase Superscript (Gibco-BRL, USA), 10 pmol (T)-primer,biotinylized at the 5′ end (sequence 5′-biotin-GGGAGGCCC(T)₁₃), 30 URNAse inhibitor from human placenta, dATP, dGTP, dCTP, dTTP (1 mM each),1× buffer of reverse transcriptase in accordance with manufacturer'srecommendations make up the reaction mixture. Removal of the primer isdone with the aid of reprecipitation using cetyltrimethylammoniumbromide [18], after which additional purification is carried out on aWizard column (Promega, USA) according to manufacturer'srecommendations. The purified preparation is precipitated with 3 volumesof ethanol using 2 μg glycogen (Boehringer-Mannheim, Germany) ascarrier.

[0031] Suspension of the oligo(dT) tail is done under the followingconditions: 37° C., 20 min, reaction volume 20 μL, with the reactionmixture containing hybrid mRNA-first chain of cDNA, 20 U terminaltransferase (Gibco-BRL, USA), 0.02 mM dGTP, 1× buffer of terminaltransferase in accordance with manufacturer's recommendations. Synthesisof the second chain of cDNA is done under the following conditions:denaturation 98° C., 1.5 min, annealing 60° C., 2 min, elongation 72°C., 20 min, reaction volume 25 μL, reaction mixture contains: hybridmRNA-cDNA, 10 pmol (C)-primer (sequence 5′-AAGGAATT(C)₁₃), dATP, dGTP,dCTP, dTTP (0.1 mM each), 1.5 U DNA polymerase Bio-Taq (Biomaster,Russia), 1× buffer of Bio-Taq in accordance with manufacturer'srecommendations. Cleavage of cDNA by restriction endonuclease is doneunder the following conditions: 37° C., 60 min, reaction volume 20 μL, 4U restriction endonuclease Sau3A (New England Biolabs, USA), 1× bufferof enzyme (according to manufacturer's recommendations). Afterhydrolysis the reaction is stopped by the addition of EDTA to 20 mM andthe 3′ and fragments of cDNA are immobilized on Streptavidin microbeads(Promega, USA) in accordance with manufacturer's recommendation. Theadaptor is added:

[0032] 1. 5′-GATCGTTTTTTGAAGCTTGGAGCCCAC-3′

[0033] 2. 3′-CAAAAAACTTCGAACCTCGGGTG-5′

[0034] and ligated at 12° C. overnight. Reamplification of the cDNAfragments with the aid of PCR is done using Bio-(T₁₃) primer and primer1 under the following conditions: denaturation 95° C., 1.2 min,annealing 55° C., 1.5 min, elongation 72° C., 3 min* reaction volume 100μL, reaction mixtures contains: 30 pmol Bio-(T) primer, 30 pmol primer1, 2.5 U DNA polymerase Bio-Taq; the mixture of deoxynucleotidetriphosphates, 0.1 mM each, 1× buffer of Bio-Taq. After 15 PCR cyclesthe fragments are immobilized on Streptavidin microbeads (Promega, USA),then the free chain is removed by treatment with 100 mM NaOH for 10 min,rinsing with a buffer of composition: 40 mM Tris-Cl, pH 7.0, 20 mMMgCl₂, 50 [?] NaCl, primer 3 (5′-GTGGGCTCCAAGCTTC) is annealed,radioactive α-³²P dATP is added and the marker is added over 5 min usingmodified DNA polymerase of phage T7 Sequenase (United StatesBiochemicals, USA), after which the mixture of dNTP is added (to 0.2 mMeach) and complete chains are finished. Cleavage of the fragments isdone successively with restriction endonucleases EcoRV, PstI, MspI, HinPI (New England Biolabs, USA). After each endonuclease treatment thefragments are collected, denatured and applied to sequesteringpolyacrylamide gel (5% acrylamide, 0.25% methylene-bisacrylamide, 7Murea, 1× TBE buffer). Separation of the fragments is done under standardconditions after which the gel is fixed in 10% acetic acid for 30 min,dried and autoradiographed. The sequences of mRNAs that aredifferentially expressed in thymus and spleen are identified by means ofa direct comparison of the sets of bands of cDNA obtained from thymusand spleen and separated in adjacent tracks. For cloning of thedifferentially expressed sequences the corresponding bands of gel arecut apart and the fragments are eluted by incubation in 150 mM NaCl, 50mM Tris-Cl, pH 8.0, 10 mM EDTA overnight. Than precipitation of thefragments is carried out with three volumes of 96% ethanol usingglycogen as carrier. The oligo(dG) tail is suspended at the 3′ end ofthe fragment using terminal transferase, as described above, and thefragment that has been processed in this way is amplified by means ofPCR using (C) primer and primer 1.

[0035] The amplified fragment is purified with the aid ofelectrophoresis in agarose gel, transferred to low melting agarose andrecovered in pure form with the aid of phenol extraction [19]. Forcloning of the fragments they are treated with restrictase Sau3A andEcoRI and ligated to plasma vector pUC18 cleaved by restrictase BamHIand EcoRI, after which they transform competent bacteria. The presenceof recombinant clones is verified with the aid of amplification ofinsertions using PCR. Verification of the specificity of the clonedfragments is done by means of hybridization of ³²P-labeled insertionsfrom clones with blots of amplified cDNA and poly (A)⁺ RNA from thecorresponding organs.

[0036] The data in FIG. 3 show that this method leads to the formationof a multitude of labeled discrete fragments of cDNAs which can beseparated in gel electrophoresis, and in comparing the pictures of theseparation of cDNAs from two different organs one can identifydifferential bands. The fragments corresponding to the differentialbands can be amplified and cloned by the method indicated in FIG. 2. Thedata given in FIG. 4 show that the cloned fragments of cDNAs are indeedmRNAs differentially expressed in two organs.

References

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1. A method of identification of differentially expressed messenger RNA(mRNA) which consists of synthesizing from sets of mRNA sets offragments of complementary DNA (cDNA), which are separated with the aidof gel electrophoresis, the separation pictures of the cDNA fromdifferent types of cells are compared and one identifies fragments withdifferential intensity of signals, and which is distinguished by thefact that, for the formation of a set of fragments, cleavage of the cDNAis carried out with the aid of restriction endonucleases.
 2. A method asin claim 1, which is distinguished by the fact that for creation of theset of fragments of cDNA one uses only those restriction fragments thatcorrespond to the 3′ or 5′ and regions of the mRNA and the fragments ofcDNA are labeled by introducing at one end a radioactive label, achemical grouping or specific sequence of nucleotides.
 3. A method as inclaim 2, which is distinguished by the fact that for creation of the setof fragments of cDNA one uses specific immobilization of and fragmentson a solid carrier.
 4. A method as in claim 3, which is distinguished bythe fact that formation of the set of fragments of cDNA is done by meansof ligation, to and fragments of cDNA that are immobilized on a solidcarrier and that correspond to the 3′ or 5′ ends of the mRNA, of adouble-chain adaptor or single-chain oligonucleotide and reamplificationof the set of fragments by means of a polymerase chain reaction.
 5. Amethod as in claim 4, which is distinguished by the fact that forequalization of the degree of representation of different sequences inthe set of fragments single or multiple removal of the most representedsequences in the set of fragments of cDNA is done by means ofhybridization of nonimmobilized chains of fragments of cDNA withcomplementary chains immobilized on a solid carrier.
 6. A method as inclaim 4 or 5, which is distinguished by the fact that, with the goal ofimproving the sensitivity and resolution of the method, the initial setof fragments of cDNA is divided into nonintersecting subsets offragments.
 7. A method as in claim 6, which is distinguished by the factthat the division of the initial set of fragments of cDNA into subsetsis done by means of immobilization of the set of fragments labeled atone end, by the other end on a solid phase and successive cleavage witha set of restriction endonucleases.
 8. A method as in claim 6, which isdistinguished by the fact that the division of the initial set intosubsets is done with the aid of several separate amplification reactionswith paired combinations of primers corresponding to and sequences thatare common for all of the fragments of cDNA, but that contain anadditional base of the 3′ and, immediately after the regions that arecommon for all the fragments of cDNA.
 9. A method as in any of claims4-8, which is distinguished by the fact that radioactive ³²P or ³³P isused as the label.
 10. A method as in any of claims 4-8, which isdistinguished by the fact that a chemical grouping is used as the labeland the separated fragments after transfer to a membrane are detectedwith the aid of a reagent that specifically binds this group and isconjugated with the enzyme, by means of registration of the enzymaticactivity of this enzyme.
 11. A method as in any of claims 4-8, which isdistinguished by the fact that the separated fragments are transferredto a membrane and after immobilization visualization of the fragments isdone by means of hybridization with a labeled oligonucleotidecorresponding to the adaptor sequence.
 12. A method as in any of claims4-8, which is distinguished by the fact that the separated fragments aretransferred from membrane and after immobilization visualization of thesubsets of separated fragments is done by means of hybridization of themembrane with a set of labeled oligonucleotides that correspond to theadaptor sequence and that carry at the 3′ and one or several additionalbases.
 13. A method as in any of claims 4-12, which is distinguished bythe fact that the fragments are separated by two-dimensional gelelectrophoresis.
 14. A method as in claim 13, which is distinguished bythe fact that fragments are separated by length in one direction oftwo-dimensional electrophoresis and by melting profile using adenaturing gradient in the other direction.
 15. A method as in claim 13,which is distinguished by the fact that single-chain fragments areseparated by length under denaturing conditions in one direction oftwo-dimensional electrophoresis and according to secondary structure innondenaturing conditions in the second direction.
 16. A method as inclaim 13, which is distinguished by the fact that fragments areseparated by length in the first direction of two-dimensionalelectrophoresis and in the switch to the second direction the fragmentsof cDNA are specifically cleaved in the gel by a set of restrictionendonucleases, after which a second separation by length is carried out.17. A method of cloning of differentially expressed mRNAs which consistsof synthesizing from a set of sequences of mRNAs from different types ofcells sets of fragments of complementary DNA (cDNA) which are separatedwith the aid of gel electrophoresis, the pictures of separation of thecDNA from different types of cells are compared, the fragments of cDNAwith different signal intensities are separated from the gel, amplifiedwith the aid of a polymerase chain reaction and cloned to a plasmid orphage vector, and which is distinguished by the fact that, for theformation of the set of fragments one carries out cleavage of the cDNAwith the aid of restriction endonucleases and uses only those fragmentsof cDNA that correspond to the 3′ or 5′ end regions of the mRNAs.Summary A method of identification of differentially express messengerRNA (mRNA) which consists of synthesizing from a set of sequences ofmRNA sets of fragments of complementary DNA (cDNA), which are separatedwith the aid of gel electrophoresis and the pictures of separation ofthe cDNA from different types of cells are compared and fragments withdifferential signal intensity are identified. For formation of the setof fragments the cDNA is cleaved with the aid of restriction nucleases.A method of cloning of differentially expressed mRNAs consists ofsynthesizing from sets of sequences of mRNAs from different types ofcells sets of fragments of complementary DNA (cDNA) which are separatedwith the aid of gel electrophoresis, the pictures of the separation ofthe cDNA from different types of cells are compared, fragments of cDNAwith different signal intensities are separated from the gel, amplifiedwith the aid of a polymerase chain reaction and cloned to a plasmid orphage vector. For the formation of the set of fragments one carries outcleavage of the cDNA with the aid of restriction endonucleases and usesonly those fragments of cDNA that correspond to the 3′ or 5′ end regionsof the mRNAs.